lncRNA NKILA对鼻咽癌细胞放射抵抗和上皮间质转化的影响及其机制

Effects of lncRNA NKILA on epithelial mesenchymal transformation and radiation resistance of nasopharyngeal carcinoma cells

目的探讨长链非编码RNA(lncRNA)NKILA对鼻咽癌细胞放射抵抗和上皮间质转化的影响及其机制。方法体外传代培养鼻咽癌细胞CNE2并构建放射抵抗细胞模型。将放射抵抗CNE2细胞随机分为空白组、NC组、转染组,转染组和NC组分别转染NKILA-mimic、NKILA-NC质粒,空白组不予转染。收集细胞,采用RT-qPCR法检测lncRNA NKILA、miR-21表达,采用MTT法检测细胞增殖能力,采用Transwell小室实验检测细胞侵袭和迁移能力,采用Western blotting法检测E-cadherin、N-cadherin表达;通过TargetScan预测lncRNA NKILA与miR-21的靶向调控关系,并通过双荧光素酶报告基因实验验证lncRNA NKILA与miR-21的靶向调控关系。取SD裸鼠10只,随机分为观察组和对照组各5只,分别于右前肢腋下注射转染lncRNA NKILA的放射抵抗CNE2细胞、单纯放射抵抗CNE2细胞,常规饲养4周处死,分离并测量移植瘤体积。结果转染组lncRNA NKILA表达、E-cadherin表达均高于空白组和NC组,miR-21表达及细胞增殖、侵袭、迁移能力和N-cadherin表达均低于空白组和NC组(P均<0.05);而空白组与NC组上述指标比较差异均无统计学意义(P均>0.05)。TargetScan预测显示,lncRNA NKILA与miR-21存在靶向结合位点;双荧光素酶报告基因实验结果显示,共转染NKILA-mimic质粒与miR-21-3′-UTR-WT的CNE2细胞荧光素酶活性低于共转染NKILA-NC质粒与miR-21-3′-UTR-WT的CNE2细胞(P<0.05),而共转染NKILA-mimic质粒、miR-21-3′-UTR-MUT与共转染NKILA-NC质粒、miR-21-3′-UTR-MUT的CNE2细胞荧光素酶活性比较差异无统计学意义(P>0.05)。观察组移植瘤体积小于对照组(P<0.05)。结论lncRNA NKILA可能通过靶向抑制miR-21表达而抑制鼻咽癌细胞的增殖、侵袭和迁移,继而抑制鼻咽癌细胞的放射抵抗和上皮间质转化。

Objective To investigate the effects of lncRNA NKILA on epithelial-mesenchymal transition and radia⁃tion resistance of nasopharyngeal carcinoma cells and its mechanism.Methods Nasopharyngeal carcinoma cells CNE2 were cultured in vitro and the radio-resistant cell models were constructed.Radio-resistant CNE2 cells were randomly divided into the blank group,NC group,and transfection group.Cells in the transfection group and NC group were trans⁃fected with NKILA-mimic and NKILA-NC plasmid,respectively;cells in the blank group were not transfected.LncRNA NKILA and miR-21 expression levels were detected by RT-qPCR,cell proliferation was detected by MTT,and cell inva⁃sion and migration were detected by Transwell assay.The expression of E-cadherin and N-cadherin proteins was detected by Western blotting.The targeted relationship between lncRNA NKILA and miR-21 was predicted by TargetScan,and the targeted relationship between lncRNA NKILA and miR-21 was verified by double luciferase reporter gene experiment.Ten SD nude mice were randomly divided into the observation group and control group,with 5 in each group.Radio-resistant CNE2 cells transfected with lncRNA NKILA and simple radio-resistant CNE2 cells were injected into the armpit of the right forelimb,respectively.The mice were killed in routine feeding for 4 weeks,and the transplanted tumor volume was separated and measured.Results The expression levels of lncRNA NKILA and E-cadherin in the transfection group were significantly higher than those in the blank group and NC group,while the expression of miR-21,cell proliferation,invasion,migration and N-cadherin protein in the transfection group were significantly lower than those in the blank group and NC group(all P<0.05).There was no significant difference in the above indexes between the blank group and the NC group(all P>0.05).TargetScan prediction showed that lncRNA NKILA had targeted binding sites with miR-21.The results of double luciferase reporter gene experiment showed that the luciferase activity of CNE2 cells co-transfected with NKILA-mimic plasmid and miR-21-3'-UTR-WT was significantly lower than that of CNE2 cells co-transfected with NKILA-NC plasmid and miR-21-3'-UTR-WT(P<0.05).There was no significant difference in the luciferase activity between CNE2 cells co-transfected with NKILA-mimic plasmid and miR-21-3'-UTR-MUT and those co-transfected with NKILA-NC plasmid and miR-21-3'-UTR-MUT(P>0.05).The transplanted tumor volume of the observation group was significantly lower than that of the control group(P<0.05).Conclusion LncRNA NKILA can inhibit the proliferation,invasion and migration of nasopharyngeal carcinoma cells through targeted inhibition of miR-21 expression,and then inhibit the radiore⁃sistance and epithelial mesenchymal transformation of nasopharyngeal carcinoma cells.